Fig 2: Astrocyte processes obtained from adult rat cerebral cortex. Negligible contamination of gliosomes, positive for the specific glial marker GFAP (A, D, G), by subcellular no-astrocytic particles. Immunofluorescent assay for synaptophysin (B), RIP (E) or integrin-aM (H) markers for nerve terminals, microglia, and oligodendrocytes, respectively. As a positive control, the immunofluorescent assay for synaptophysin (J) was performed on cerebral cortical synaptosomes scarcely contaminated by subcellular GFAP-positive particles (K). Bars (C, F, I, L) represent the percent of positive particles (% ± SEM of positive particles counted in three to five no-overlapping fields from n = 3 different preparations): GFAP (C, F, I, solid bars; L, empty bar), synaptophysin (C empty bar; L solid bar; L, empty bar), or RIP or integrin-aM (F or I, respectively; empty bar). Scale bars are indicated in the figures. Western blot analysis of gliosomes and synaptosomes (M, N). The absence of cross-contamination of the astrocyte processes and nerve terminals is shown (M): MAP2, ß III tubulin, and GFAP proteins were used as selective markers for the synaptosome or gliosome preparations. Presence of the astrocytic markers GFAP and ezrin, and of the exosome markers Alix and Tsg101 in the gliosomes (N). Electron microscopy image of a cortical astrocyte processes. A single gliosome is shown containing vesicles scattered in the cytoplasm and a multivesicular body (O). Scale bars: 200 nm. Schematic of a perfusion unit of the apparatus allowing recovery of extracellular vesicles (exosomes) released from the processes during perfusion (P). For other experimental details, see Materials and Methods.

Fig 3: ALIX and the ESCRT-I recruitment factor TSG101 are corecruited with ALG-2 to damaged lysosomes. (A) U2OS cells were treated with either DMSO (Upper row) or LLOME (Lower row) for 15 min and then immunostained for ALG-2 (green), ALIX (magenta), TSG101 (yellow), and Gal-3 (cyan). Relatively large colocalized puncta of ALG-2, ALIX, and TSG101 along with Gal-3 were observed after LLOME treatment. The regions encapsulated within the white boxes have been enlarged and the individual protein channels are shown on the Right. Images are representative of five independent replicates. (B) The area of overlapping compartments increased dramatically upon LLOME treatment compared to the DMSO control (n = 75 cells for DMSO, 64 cells for LLOME). The circles on the bar charts represent independent data points and the data are shown as mean ± SD (vertical line). P = 0.0002 (***). (Scale bar, 5 µm.)

Fig 4: Complete reconstitution of Ca2+-triggered endolysosomal membrane repair machinery. (A) Fluorescently labeled ALIX (Cy3; 100 nM), CHMP4B (Atto 488; 10 nM), and VPS4B (LD 655; 100 nM) along with ALG-2 (dark; 200 nM), CHMP2A (dark; 100 nM), and CHMP3 (dark; 100 nM) were mixed with 30% DOPS containing GUVs and imaged. The observance of colocalized puncta of the labeled proteins on the periphery of GUVs confirms the recruitment of the entire endolysosomal membrane repair machinery. (B) In the absence of CHMP4B, labeled VPS4B was not recruited to the ALIX puncta on the membrane. (C) The bar chart depicting the colocalization % of the puncta in two different fluorescently labeled protein channels. At least 100 GUVs from three independent experiments were analyzed for each set of conditions plotted in the bar chart. The circles on the bar plots represent independent data points and the data are shown as mean ± SD (vertical line). P = 0.0003 (***). (Scale bar, 10 µm.)

Fig 5: Intercellular sgRNA exchange is EV-mediated.a Schematic of EV isolation workflow and analysis. Conditioned medium is isolated from sgRNA+ MDA-MB-231 donor cells, after which cell debris is pelleted through centrifugation. Conditioned medium is then concentrated by tangential flow filtration, followed by size exclusion chromatography-mediated EV isolation. EVs are then characterized, and used for EV addition experiments in Stoplight+spCas9+ reporter cells. b Nanosight Nanoparticle Tracking Analysis (NTA) showing the size distribution of the isolated EVs (mode = 81 ± 3 nm). c Western blot analysis of EVs and cell lysates for common EV markers (CD9, CD63, ALIX, Flot-1, and TSG101), and EV-negative markers Calnexin and H2B. Representative images as observed in three independent experiments. d Electron microscopy analysis of isolated EVs. Scale bar represents 100 nm. Representative image as observed in 12 separate fields. e An EV RNase protection assay, followed by RT-PCR sgRNAs, shows that sgRNAs are only susceptible to RNase-mediated degradation in the presence of the membrane-disrupting anionic surfactant SDS. Representative data as observed in three independent experiments. f Confocal microscopy images of HEK293T reporter cells (red) that have taken up MDA-MB-231-derived PKH67-labeled EVs (green). Representative image as observed in seven randomly selected fields. g, h EV-mediated activation of the CROSS-FIRE platform using EVs isolated from sgRNA+ MDA-MB-231 donor cells. EVs were added every 72 h for 12 additions with an average dose of 1.1e11 ± 4.9e10 EVs per addition. Cells were analyzed by fluorescence microscopy (g) and flow cytometry (h). Scale bar represents 200 µm. Means + SD, n = 3 biological replicates, Tukey’s multiple comparison test. i EVs from sgRNA+ MDA-MB-231 donor cells activate Stoplight+ spCas9+ HEK293T reporter cells in a dose-dependent manner. EVs were added every 72 h for nine additions with an average concentration of 2.2e10 ± 1e10 (20 µl) or 2.2e11 ± 1e11 (200 µl). Means + SD, n = 3 biological replicates, Tukey’s multiple comparison test. *p < 0.05.